The present invention relates to promoters, enhancers and other regulatory elements that direct expression within smooth muscle cells (xe2x80x9cSMCxe2x80x9d). In particular, it relates to compositions comprising nucleotide sequences from the 5xe2x80x2 regulatory region and the first intron, and transcriptionally active fragments thereof, that control expression of a smooth muscle xcex1-actin (xe2x80x9cSM xcex1-Axe2x80x9d). Specifically provided are expression vectors, host cells and transgenic animals wherein an SM xcex1-A regulatory region is capable of controlling expression of a heterologous gene, over-expressing an endogenous SMC gene or an inhibitor of a pathological process or knocking out expression of a specific gene believed to be important for an SM-related disease in SMC. The invention also relates to methods for using said vectors, cells and animals for screening candidate molecules for agonists and antagonists of disorders involving SMC.
The present invention further relates to compositions and methods for modulating expression of compounds within SMC. The invention further relates to screening compounds that modulate expression within SMC. Methods for using molecules and compounds identified by the screening assays for therapeutic treatments also are provided.
Somatic cell gene therapy is a strategy in which a nucleic acid, typically in the form of DNA, is administered to alter the genetic repertoire of target cells for therapeutic purposes. Although research in experimental gene therapy is a relatively young field, major advances have been made during the last decade. (Arai, Y., et al., 1997, Orthopaedic Research Society, 22:341). The potential of somatic cell gene therapy to treat human diseases has caught the imagination of numerous scientists, mainly because of two recent technologic advancements. Firstly, there are now numerous viral and non-viral gene therapy vectors that can efficiently transfer and express genes in experimental animals in vivo. Secondly, increasing support for the human genome project will allow for the identity and sequence of the estimated 80,000 genes comprising the human genome in the very near future.
Gene therapy was originally conceived of as a specific gene replacement therapy for correction of heritable defects to deliver functionally active therapeutic genes into targeted cells. Initial efforts toward somatic gene therapy relied on indirect means of introducing genes into tissues, called ex vivo gene therapy, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes and re-implanted into the body (xe2x80x9cautologous cell transferxe2x80x9d). A variety of transfection techniques are currently available and used to transfer DNA in vitro into cells; including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer or transduction with recombinant viral vectors. Such ex vivo treatment protocols have been proposed to transfer DNA into a variety of different cell types including epithelial cells (U.S. Pat. No. 4,868,116; Morgan and Mulligan WO87/00201; Morgan et al., 1987, Science 237:1476-1479; Morgan and Mulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345), hepatocytes (WO89/07136; Wolff et al., 1987, Proc. Natl. Acad. Sci. USA 84:3344-3348; Ledley et al., 1987 Proc. Natl. Acad. Sci. 84:5335-5339; Wilson and Mulligan, WO89/07136; Wilson et al., 1990, Proc. Natl. Acad. Sci. 87:8437-8441), fibroblasts (Palmer et al., 1987, Proc. Natl. Acad. Sci. USA 84:1055-1059; Anson et al., 1987, Mol. Biol. Med. 4:11-20; Rosenberg et al., 1988, Science 242:1575-1578; Naughton and Naughton, U.S. Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No. 5,399,346; Blaese, R. M. et al., 1995, Science 270:475-480) and hematopoietic stem cells (Lim, B. et al. 1989, Proc. Natl. Acad. Sci. USA 86:8892-8896; Anderson et al., U.S. Pat. No. 5,399,346).
Direct in vivo gene transfer recently has been attempted with formulations of DNA trapped in liposomes (Ledley et al., 1987, J. Pediatrics 110:1), in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1068) and DNA coupled to a polylysine-glycoprotein carrier complex. In addition, xe2x80x9cgene gunsxe2x80x9d have been used for gene delivery into cells (Australian Patent No. 9068389). It even has been speculated that naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions for injection into interstitial spaces for transfer of DNA into cells (Felgner, WO90/11092).
Numerous clinical trials utilizing gene therapy techniques are underway for such diverse diseases as cystic fibrosis and cancer. The promise of this therapeutic approach for dramatically improving the practice of medicine has been supported widely, although there still are many hurdles that need to be passed before this technology can be used successfully in the clinical setting.
Perhaps, one of the greatest problems associated with currently devised gene therapies, whether ex vivo or in vivo, is the inability to control expression of a target gene and to limit expression of the target gene to the cell type or types needed to achieve a beneficial therapeutic effect.
Smooth muscle cells, often termed the most primitive type of muscle cell because they most resemble non-muscle cells, are called xe2x80x9csmoothxe2x80x9d because they contain no striations, unlike skeletal and cardiac muscle cells. Smooth muscle cells aggregate to form smooth muscle (xe2x80x9cSMxe2x80x9d) which constitutes the contractile portion of the stomach, intestine and uterus, the walls of arteries, the ducts of secretory glands and many other regions in which slow and sustained contractions are needed.
Abnormal gene expression in SMC plays a major role in numerous diseases including, but not limited to, atherosclerosis, coronary artery disease, hypertension, stroke, asthma and multiple gastrointestinal, urogenital and reproductive disorders. These diseases are the leading causes of morbidity and mortality in Western Societies, and account for billions of dollars in health care costs in the United States alone each year.
In recent years, the understanding of muscle differentiation has been enhanced greatly with the identification of several key cis-elements and trans-factors that regulate expression of muscle-specific genes. Firulli A. B. et al., 1997, Trends in Genetics, 13:364-369; Sartorelli V. et al., 1993, Circ. Res., 72:925-931. However, the elucidation of transcriptional pathways that govern muscle differentiation has been restricted primarily to skeletal and cardiac muscle. Currently, no transcription factors have yet been identified that direct SM-specific gene expression, or SMC myogenesis. Owens G. K., 1995, Physiol. Rev., 75:487-517. Unlike skeletal and cardiac myocytes, SMC do not undergo terminal differentiation. Furthermore, they exhibit a high degree of phenotypic plasticity, both in culture and in vivo. Owens G. K., 1995, Physiol. Rev., 75:487-517; Schwartz S. M. et al., 1990, Physiol. Rev., 70:1177-1209. Phenotypic plasticity is particularly striking when SMC located in the media of normal vessels are compared to SMC located in intimal lesions resulting from vascular injury or atherosclerotic disease. Schwartz S. M., 1990, Physiol. Rev., 70:1177-1209; Ross R., 1993, Nature, 362:801-809; Kocher 0. et al., 1991, Lab. Invest., 65:459-470; Kocher O. et al., 1986, Hum. Pathol., 17:875-880. Major modifications include decreased expression of SM isoforms of contractile proteins, altered growth regulatory properties, increased matrix production, abnormal lipid metabolism and decreased contractility. Owens G. K., 1995, Physiol. Rev., 75:487-517. The process by which SMC undergo such changes is referred to as xe2x80x9cphenotypic modulationxe2x80x9d. Chamley-Campbell J. H. et al., 1981, Atherosclerosis, 40:347-357. Importantly, these alterations in expression patterns of SMC protein cannot simply be viewed as a consequence of vascular disease, but rather, are likely to contribute to progression of the disease.
A key to understanding SMC differentiation is to identify transcriptional mechanisms that control expression of genes that are selective or specific for differentiated SMC and that are required for its principal differentiated function, contraction. Currently, studies are ongoing in which the expression of the contractile proteins SM xcex1-A (Shimizu R. T. et al., 1995, J. Biol. Chem., 270:7631-7643; Blank R. S. et al., 1992, J. Biol. Chem., 267:984-989) and SM myosin heavy chain (SM-MHC)(White S. L. et al., 1996, J. Biol. Chem., 271:15008-15017; Katoh Y. et al., 1994, J. Biol. Chem., 269:30538-30545; Wantanabe M. et al., 1996, Circ. Res., 78:978-989; Kallmeier R. C. et al., 1995, J. Biol. Chem., 270:30949-30957; Madsen C. S. et al., 1997, J. Biol. Chem., 272:6332-6340; Madsen C. S. et al., 1997, J. Biol. Chem., 272:29842-29851), as well as a variety of proteins implicated in control of contraction including SM22xcex1(Li L. et al., 1996, J. Cell. Biol., 132:849-859; Kim S. et al., 1997, Mol. Cell. Biol., 17:2266-2278), h1-calponin (Miano J. M. et al., 1996, J. Biol. Chem. 271:7095-7103), h-caldesmon (Yano H. et al., 1994, Biochem. Biophys. Res. Commun. 201:618-626), telokin (Herring B. P. et al., 1996, Am. J. Physiol., 270:C1656-C1665) and desmin (Bolmont C. et al., 1990, J. Submicrosc. Cytol. Pathol., 22:117-122) are being examined.
Recently, several cis elements and trans acting factors have been described that regulate muscle-specific gene expression in skeletal and cardiac muscle and are required for the terminal differentiation of these muscle cell types. In contrast, the mechanisms regulating SMC differentiation are only poorly understood, and to date, no transcription factors have been identified that direct SMC-specific gene expression. Because SMC maturation and differentiation are required for the full development of arteries and veins during angiogenesis and vasculogenesis, the identification of the molecular mechanisms that control SMC differentiation are important for an understanding of these processes that occur not only during development, but also under pathologic conditions. Furthermore, it may lead to a better understanding of SMC phenotypic modulation that has been shown to contribute to atherosclerosis and restenosis following balloon angioplasty (Ross R, et al., N. Engl J Med. 1976;295:369-377; Schwartz SM, et al.; Prog Cardiovasc Dis. 1984;26:355-372).
One example of a protein which is required for contractile functions of SMC is SM xcex1-actin, which makes up 40% of total SMC protein. Not only is it clearly required for the contractile function of SMC, but it also is the first SMC differentiation marker to appear during development (Duband J L, et al.; Differentiation; 1993;55:1-11). Although SM xcex1-A is transiently expressed in the myocardium and skeletal muscle in the developing embryo, and in myofibroblasts during wound healing, SM xcex1-A expression in adult animals is highly restricted to SMC or SM-like cells (Darby I, et al.; Lab Invest.; 1990;63:21-29; Woodcock-Mitchell J, et. al.; Differentiation; 1988;39:161-166).
Transcriptional regulation of various SMC genes has been analyzed extensively in cultured SMC and several functional cis-elements have been identified. White S. L. et al., 1996, J. Biol. Chem., 271:15008-15017; Katoh Y. et al., 1994, J. Biol. Chem. 269:30538-30545; Wantanabe M. et al., 1996, Circ. Res., 78:978-989; Kallmeier R. C. et al., 1995, J. Biol. Chem., 270:30949-30957; Madsen C. S. et al., 1997, J. Biol. Chem., 272:6332-6340; Madsen C. S. et al., 1997, J. Biol. Chem., 272:29842-29851. However, because differentiation of SMC is known to be dependent on many local environmental cues that cannot be completely reproduced in vitro, cultured SMC are known to be phenotypically modified as compared to their in vivo counterparts (Owens G. K., 1995, Physiol. Rev., 75:487-517; Chamley-Campbell J. H. et al., 1981, Atherosclerosis, 40:347-357). As such, certain limitations exist regarding the usefulness of cultured SMC in defining transcriptional programs that occur during normal SMC differentiation and maturation within the animal.
One example of a transcriptional regulatory element that has been implicated in the transcriptional control of various SMC genes is the CArG element. The CArG element was first described as the core sequence of the serum response element (SRE) within early response genes such as c-fos, but also has been shown to be required for the activity of many muscle-specific gene promoters (Gustafson T A, et al., Mol. Cell Biol.; 1988;8:4110-4119; Chow K, et al., Mol. Cell Biol., 1990;10:528-538; Papadopoulos N, et al., Mol. Cell Biol., 1993;13:6907-6918; Mohun T J, et al., EMBO J., 1989;8:1153-1161; Lee, T, et al., Mol. Cell Biol., 1991;11:5090-5100). Of interest, nearly all of the SMC differentiation marker genes characterized to date, including SM myosin heavy chain (SM MHC), caldesmon and telokin, contain two or more CArG elements that are required for maximal expression in cultured SMC (Shimizu R T, et al., J. Biol Chem., 1995;270:7631-7643; Madsen C S, et al., J Biol Chem., 1997;272:6332-6340; Li L, et al., J. Cell Biol., 1996;132:849-859; Herring B P, et al., Am. J. Physiol., 1997;272:C1394-C1404; White S L, et al., J. Biol. Chem., 1996;271:15008-15017; Zilberman A, et al., Circ. Res., 1998;82:566-575). In addition, it previously has been reported that a conserved CArG element in the SM-22 promoter is required for the arterial expression of a Lac Z transgene in the mouse (Kim S, et al., Mol. Cell Biol., 1997;17:2266-2278; Li L, et al., Dev. Biol., 1997;187:311-321). Electrophoretic mobility supershift studies demonstrated that the SM xcex1-A CArG elements, like the SRE, bind serum response factor (Shimizu R T, et al., J. Biol Chem., 1995;270:7631-7643). Although recent evidence suggests that muscle derived tissues express higher levels of SRF than nonmuscle tissues (Li L, et al., Dev. Biol., 1997;187:311-321), SRF is thought to be ubiquitously expressed, and a critical yet presently unresolved question remains as to the mechanism of CArG-dependent regulation of SMC-specific gene expression.
It is now well established that SMC differentiation is dependent upon a large number of local environmental cues including extracellular matrix interactions, local production of growth factors and mechanical stresses that cannot be accurately reproduced in culture (Owens G. K., Physiol. Rev., 1995;75:487-517; Chamley-Champbell J H, et al., Atherosclerosis., 1981;40:347-357). Moreover, recent studies have provided clear evidence that gene regulation in SMC culture systems does not always represent regulation in vivo. Li L, et al., Dev. Biol. 1997;187:311-321; Madsen C S, et al., Circ. Res., 1998;82:908-917. As such, when studying SMC differentiation, it is critical that regulatory pathways initially identified in cultured SMC are tested in vivo through the use of transgenic animals. For example, analysis of SM-22 and SM MHC gene expression in transgenic mice has demonstrated that expression of SMC-marker genes is complex and may involve xe2x80x9cregulatory cassettesxe2x80x9d that drive expression within some, but not all, SM tissues (Li L, et al., J. Cell Biol., 1996;132:849-859; Kim S, et al., Mol. Cell Biol., 1997;17:2266-2278). As such, transgenic studies also are critical for detecting possible heterogeneity in SMC gene regulation.
Currently, no studies have reported the complete characterization of regulatory regions required for driving in vivo expression of SM xcex1-A during development and maturation. Although Wang et al. (Wang J, et al., J. Clin Invest., 1997;100:1425-1439) recently reported that an SM xcex1-A promoter containing 1,100 bp of 5xe2x80x2 promoter and the entire first intron could drive expression of an IGF-1 transgene in many SM tissues, there studies were restricted to analysis in adult animals and focused on examination of the effects of IGF-1 overexpression in SMC and not on the characterization of the promoter regions required for SMC-specific expression. This deficiency of Wang et al. is critical since the SM xcex1-A gene is known to be expressed by all three muscle types during development. Moreover, it is highly likely that over-expression of the biologically active substance IGF-1 in the studies by Wang et al. resulted in feedback alterations in the activity of the SM xcex1-A promoter since there is extensive evidence that IGF-1 alters SMC function (Clemmons et al., J Cell Physiol, 145:129-135, 1990). As such, it is unclear whether the expression patterns reported by Wang et al. are truly representative of the inherent activity of the SM xcex1-A promoter, as opposed to being artifactually influenced by over-expression of IGF-1.
The current invention provides the major advance of identifying molecular elements that confer SMC-specific transcription in vivo during normal development and during various disease states involving SMC-specific gene expression. More specifically, the instant invention provides, for the first time, inter alia, the identification of sufficient regions of the SM xcex1-A gene to direct SMC-specific expression, both in vitro in cultured SMC, and in vivo in transgenic animals.
The invention disclosed herein provides a model for SMC-specific gene transcription. The invention is based in part on the functional characterization described herein of an SM xcex1-A regulatory region, which is the first SMC-specific regulatory region found to be active only in SMC.
The present invention provides compositions and methods for screening compounds that modulate expression within SMC. In particular, it provides compositions comprising nucleotides from the rat SM xcex1-A promoter and first intron, and transcriptionally active fragments thereof, as well as nucleic acids that hybridize under highly stringent conditions to such nucleotides, that control the expression of an SMC-specific gene. Specifically provided are expression vectors comprising the SM xcex1-A regulatory region, and transcriptionally active fragments thereof, operably associated to a heterologous reporter gene, e.g., LacZ, and host cells and transgenic animals containing such vectors. The invention also provides methods for using such vectors, cells and animals for screening candidate molecules for agonists and antagonists of SMC-related disorders. Methods for using molecules and compounds identified by the screening assays for therapeutic treatments also are provided.
For example, and not by way of limitation, a composition comprising a reporter gene is operatively linked to an SMC-specific regulatory sequence, herein called the SM xcex1-A regulatory region. The SM xcex1-A driven reporter gene is expressed as a transgene in animals. The transgenic animal, and cells derived from the SMC of such transgenic animal, can be used to screen compounds for candidates useful for modulating SMC-related disorders. Without being bound by any particular theory, such compounds are likely to interfere with the function of trans-acting factors, such as transcription factors, cis-acting elements, such as promoters and enhancers, as well as any class of post-transcriptional, translational or post-translational compounds involved in SMC-related disorders. As such, they are powerful candidates for treatment of such disorders, including, but not limited to, coronary artery disease, hypertension, stroke, asthma and multiple gastrointestinal, urogenital and reproductive disorders.
In one embodiment, the invention provides methods for high throughput screening of compounds that modulate specific expression of genes within SMC. In this aspect of the invention, cells from SM-tissues are removed from the transgenic animal and cultured in vitro. The expression of the reporter gene is used to monitor SMC-specific gene activity. In a specific embodiment, LacZ is the reporter gene. Compounds identified by this method can be tested further for their effect on SMC-related disorders in normal animals.
In another embodiment, the transgenic animal models of the invention can be used for in vivo screening to test the mechanism of action of candidate drugs for their effect on SMC-related disorders. Specifically, the effects of the drugs on SMC-related disorders including, but not limited to, coronary artery disease, hypertension, stroke, asthma and multiple gastrointestinal, urogenital and reproductive disorders, can be assayed.
In another embodiment, a gene therapy method for treating and/or preventing SMC-related disorders is provided. Smooth muscle xcex1-A regulatory sequences are used to drive SMC-specific expression of therapeutic molecules and introduced in the SMC. The method comprises introducing an SM xcex1-A regulatory sequence operatively associated with a nucleic acid encoding a therapeutic molecule into SMC. In one embodiment, the invention provides a preventative gene therapy method comprising introducing an SM xcex1-A regulatory sequence operatively associated with a nucleic acid encoding a therapeutic molecule into SMC to delay and/or prevent an SMC-related disorder. In a specific embodiment, the invention provides a gene therapy method for treatment of cancer or other proliferative disorder involving SMC. The SM xcex1-A regulatory sequence is used to direct the expression of one or more proteins specifically in the SM-tumor cells of a patient.
The invention further provides methods for screening for novel transcription factors that modulate the SM xcex1-A regulatory sequence. Such novel transcription factors identified by this method can be used as targets for treating SMC-related disorders.